U.S. patent application number 10/220970 was filed with the patent office on 2003-08-07 for reaction system for thermal cycling.
Invention is credited to Lee, Martin Alan, Squirrell, David James.
Application Number | 20030148503 10/220970 |
Document ID | / |
Family ID | 9887102 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148503 |
Kind Code |
A1 |
Squirrell, David James ; et
al. |
August 7, 2003 |
Reaction system for thermal cycling
Abstract
Method and apparatus for carrying out a thermal cycling
reaction, wherein a succession of samples is conveyed through a
series of sequentially arranged temperature control sites, each of
the sites comprising means for supplying an electric current to, or
inducing an electric current in, sample-containing vessels passing
through it so as to induce temperature changes in the samples. Also
provided is a sample support and its production, the support
comprising a succession of sample vessels arranged sequentially one
behind the next, preferably in the form of a linked chain, the
support comprising an electrically conducting, preferably plastics,
material which beats when an electric current passes through
it.
Inventors: |
Squirrell, David James;
(Salisbury, GB) ; Lee, Martin Alan; (Salisbury,
GB) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
9887102 |
Appl. No.: |
10/220970 |
Filed: |
January 10, 2003 |
PCT Filed: |
March 7, 2001 |
PCT NO: |
PCT/GB01/00988 |
Current U.S.
Class: |
435/287.2 ;
435/287.3; 435/303.1; 435/91.2 |
Current CPC
Class: |
B01L 7/52 20130101 |
Class at
Publication: |
435/287.2 ;
435/287.3; 435/303.1; 435/91.2 |
International
Class: |
C12M 001/34; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2000 |
GB |
0005434.6 |
Claims
1. Apparatus for carrying out a thermal cycling reaction,
comprising a series of sequentially arranged temperature control
sites and conveyor means for conveying a succession of samples
through then, each of the sites comprising means for supplying an
electric current to, or inducing an electric current in, a
sample-containing vessel passing through them so as to induce
temperature changes in the sample.
2. Apparatus according to claim 1, wherein the conveyor means is at
least partially automated.
3. Apparatus according to claim 1 or claim 2, wherein the
processing sites are arranged in a linear fashion.
4. Apparatus according to any one of the preceding claims,
comprising more than four sequentially arranged temperature control
sites.
5. Apparatus according to any one of the preceding claims, wherein
each temperature control site supplies or induces a constant level
of current in all sample vessels passing through it.
6. Apparatus according to any one of the preceding claims, wherein
at each temperature control site current is supplied to sample
vessels via electrical contacts incorporated in the conveyor
means.
7. Apparatus according to any one of the preceding claims,
comprising one or more additional processing sites.
8. Apparatus according to claim 7, comprising, at one or more of
the additional processing sites, monitoring means for monitoring
the composition of samples passing through the site and/or the
progress of reactions occurring in the samples.
9. Apparatus according to claim 8, wherein the monitoring means
comprises means for detecting the absorption or emission of
radiation by samples passing through the site, and/or means for
stimulating such emission.
10. Apparatus according to any one of claims 7 to 9, comprising, at
one or more of the additional processing sites, loading means for
loading reagents into a succession of sample vessels passing
through the site.
11. Apparatus according to any one of claims 7 to 9, comprising, at
one or more of the additional processing sites, means for sealing
shut sample-containing vessels passing through the site.
12. Apparatus according to any one of the preceding claims,
comprising, upstream of the temperature control sites, means for
producing a sample support which comprises a succession of separate
sample vessels arranged sequentially one behind the next, and means
for conveying the so-produced sample support to the temperature
control sites.
13. Apparatus for carrying out a thermal cycling reaction, the
apparatus being substantially as herein described with reference to
the accompanying illustrative drawings.
14. A sample support for use with apparatus according to any one of
the preceding claims, the support comprising a succession of sample
vessels arranged sequentially one behind the next and being adapted
to be conveyed continuously through a series of processing sites,
the support comprising an electrically conducting material which
heats when an electric current passes through it.
15. A sample support according to claim 14, wherein each sample
vessel incorporates a separate element made from an electrically
conducting material which emits heat when an electric current
passes through it.
16. A sample support according to claim 14 or claim 15, wherein the
electrically conducting material is a plastics material.
17. A sample support according to claim 16, wherein the
electrically conducting plastics material is a polymer loaded with
an electrically conducting material.
18. A sample support according to claim 17, wherein the
electrically conducting material is either carbon or a metal.
19. A sample support according to any one of claims 14 to 18,
wherein the sample vessels are in a linear, or substantially
linear, arrangement.
20. A sample support according to any one of claims 14 to 19,
comprising a strip of a flexible material, on which a succession of
sample vessels is mounted or in which a succession of sample
vessels is formed.
21. A sample support according to claim 20, wherein the flexible
material is an electrically conducting plastics material.
22. A sample support according to claim 20, wherein the flexible
material incorporates a layer of an electrically conducting
plastics material.
23. A sample support according to claim 21 or claim 22, comprising
a succession of electrically isolatable regions corresponding to
the positions of individual sample vessels, to allow for
independent temperature control for each of the vessels.
24. A sample support according to any one of claims 20 to 23,
wherein the sample vessels are formed in the flexible material.
25. A sample support according to any one of claims 14 to 19,
comprising a series of reaction units, each of which provides one
or more sample vessels, and which are linked together as a chain so
as to be conveyable sequentially through the processing sites.
26. A sample support according to any one of claims 14 to 25, which
is designed to be disposable after use, or wherein the sample
vessels or reaction units which it comprises are designed to be
disposable after use.
27. A sample support according to any one of claims 14 to 26,
additionally comprising electrical contacts to facilitate the
supply of electric current to the conducting material as the
support passes through an appropriate processing site.
28. A sample support according to any one of claims 14 to 27,
wherein at least a portion of each sample vessel is transparent or
translucent, so as to allow monitoring of the composition of a
sample contained in the vessel and/or the progress of a reaction
occurring in the sample.
29. A sample support according to any one of claims 14 to 28,
comprising more than three sequentially arranged sample
vessels.
30. A sample support according to claim 29, comprising ten or more
sequentially arranged sample vessels.
31. A sample support according to any one of claims 14 to 30,
wherein the sample vessels are arranged in an array.
32. A sample support according to any one of claims 14 to 31,
wherein one or more of the sample vessels is pre-loaded with one or
more reagents.
33. A sample support according to any one of claims 14 to 32, in
the form of a roll.
34. A sample support according to any one of claims 14 to 32, in
the form of a fanned stack.
35. A sample support according to any one of claims 14 to 34,
comprising engageable driving means via which it may be driven
through a succession of processing sites.
36. A sample support substantially as herein described with
reference to the accompanying illustrative drawings.
37. A method for carrying out a thermal cycling reaction, which
involves using apparatus according to any one of claims 1 to 13,
and/or a sample support according to any one of claims 14 to 36, to
convey a succession of samples through a series of sequentially
arranged temperature control sites, at each of which sites an
electric current is supplied to, or induced in, sample-containing
vessels passing through the site so as to induce temperature
changes in the samples.
38. A method according to claim 37, wherein the succession of
samples is conveyed continuously through the temperature control
sites.
39. A method according to claim 37 or claim 38, wherein the
reaction is part of an amplification reaction.
40. A method according to claim 39, wherein the reaction is part of
a PCR reaction.
41. A method according to any one of claims 37 to 40, wherein the
samples are additionally conveyed through one or more processing
sites at which the composition of the samples, and/or the progress
of reactions occurring in the samples is monitored.
42. A method according to any one of claims 37 to 41, wherein
sample vessels provided on or in a sample support are conveyed
through a processing site at which they are loaded with samples
and/or reagents.
43. A method according to any one of claims 37 to 42, which is at
least partially automated.
44. A method for carrying out a thermal cycling reaction, the
method being substantially as herein described with reference to
the accompanying illustrative drawings.
45. A method for producing a sample support according to claim 14
or any claim dependent thereon, the method comprising forming a
succession of reaction wells in a flexible strip comprising an
electrically conducting material which heats when an electric
current passes through it.
46. A method according to claim 45, wherein the electrically
conducting material is a plastics material.
47. A method according to claim 45 or claim 46, additionally
involving providing one or more of the reaction wells with one or
more appropriately positioned electrical contacts.
48. A method according to any one of claims 45 to 47, which
additionally includes pre-loading one or more of the reaction wells
with a desired reagent or reagents.
49. A method for producing a sample support according to claim 14
or any claim dependent thereon, the method being substantially as
herein described with reference to the accompanying illustrative
drawings.
50. A method according to any one of claims 37 to 44, additionally
incorporating a method according to any one of claims 45 to 49.
51. Apparatus according to any one of claims 1 to 13, in
combination with a sample support according to any one of claims 14
to 36.
52. Use of a sample support according to any one of claims 14 to 36
in a method according to any one of claims 37 to 44.
53. A method for the online monitoring of conditions in an
environment of interest, the method involving extracting samples
continually from the environment and subjecting each of the samples
successively, using a method according to any one of claims 37 to
44 or 50, to a diagnostic process involving thermal cycling.
Description
[0001] The present invention relates to methods and apparatus for
carrying out thermal cycling reactions, for instance those
necessary during an amplification reaction, in particular the
polymerase chain reaction (PCR).
[0002] Subjecting samples to thermal cycling, as is necessary for
the PCR technique, involves a series of discrete and sequential
heating and cooling steps, the speed and efficiency of which are
limited by the thermal properties of the sample containers. New
forms of container with improved thermal conductivity have helped
towards solving such problems, but there is still inevitably a time
lag during each cycle whilst the container and sample are heated or
cooled to the correct temperature.
[0003] To improve the overall efficiency of such techniques, it has
become customary to "batch process" a plurality of samples at a
time, and many forms of reaction unit are available in which an
array of samples may be held so as to be subjected together to each
processing step.
[0004] An alternative apparatus, known as the "Robo-Cycler" (trade
mark), conveys sample batches between four distinct processing
sites in an approximately circular arrangement. Each site is
maintained at a different temperature, in order to achieve thermal
cycling of the samples. The number of processing steps is limited
in this case.
[0005] The applicants have devised methods and apparatus,
embodiments of which can improve on the speed, efficiency and
versatility of such processes and which can facilitate their
automation.
[0006] According to a first aspect of the present invention there
is provided apparatus for carrying out a thermal cycling reaction,
the apparatus comprising a series of sequentially arranged
temperature control sites and conveyor means for conveying a
succession of samples through them, each of the sites comprising
means for supplying an electric current to, or inducing an electric
current in, a sample-containing vessel passing through them so as
to induce temperature changes in the sample.
[0007] The use of an electric current to cause a temperature change
in a sample can preferably be achieved by incorporating into a
vessel containing the sample an element formed from an electrically
conducting material which heats when electric current passes
through it. This allows the sample temperature to be readily
controlled, ideally separately from that of adjacent samples in the
succession, by a series of relatively simple electrical sources
located at the temperature control sites of the apparatus. Such
sources are generally less cumbersome than conventional heating
means such as heating blocks, and a large number of them can more
easily be arranged in a desired sequence for the succession of
samples to pass through. Each sample effectively carries its own
heating means with it; the temperature control sites need only
provide an appropriate source of electrical power and associated
controls. At each site an appropriate current may be applied to
achieve a desired temperature change, following which the sample
may progress to another site at which a different current may be
applied, whilst an adjacent sample in the succession is being
subjected, separately, to a similar sequence of thermal
changes.
[0008] Thus, typically, temperature changes may be induced in a
sample by moving it between successive temperature control sites.
This means that the apparatus of the invention is particularly well
suited to the sequential, effectively continuous, processing of any
desired number of samples. It is also well suited to automation,
the main controls necessary to effect thermal cycling being over
the conveyor means and the electrical current sources at the
temperature control sites.
[0009] In the context of the present invention, a "continuous"
method means one which is continuous throughout its duration, as
opposed to a purely batch method. In practice the apparatus can be
used to process a relatively large number of samples in continuous
succession (in other words, by a "semi-batch" process).
[0010] Thus, instead of (as in the prior art) a batch of samples
being either (a) heated or cooled at a first site and then conveyed
together to a second site for another heating or cooling step, or
(b) thermally cycled as a stationary batch at a single location,
all samples can be moved successively through each such site,
suitably in the form of a "chain" of samples progressing
sequentially one behind the other. Ideally, each sample reaches a
particular temperature control site at a different time to its
adjacent sample(s) in the succession. Each may therefore be
processed separately from (for instance, it may at any given time
be at a different temperature to) its adjacent sample(s).
[0011] The succession of samples which can be processed using the
apparatus is preferably linear, or substantially so, in
arrangement, or at least non-circular. The conveyor means may be
arranged so as to convey the samples continuously through the
temperature control sites, again preferably in a linear fashion,
and is preferably operable automatically or at least partially
so.
[0012] The temperature control sites of the apparatus are
preferably also arranged in a linear succession, although they need
not be in a straight line. There are preferably more than 4 of
them, more preferably more than 6, most preferably more than 10 or
16 or 20 or 50 or 100. Typically the apparatus may include up to
100, 150 or 200 temperature control sites.
[0013] At the temperature control sites, conventional equipment may
be used to cause the necessary electrical effect. Current may be
supplied, for instance, via appropriately positioned electrical
contacts which can contact complementary parts of a sample vessel
as it passes through the site. These contacts may be incorporated
in the conveyor means (for instance, a series of rollers) by which
the samples are driven through the temperature control sites.
Alternatively, a magnetic field may be used at a site to induce an
electric current in a sample vessel.
[0014] Each such site may be maintained to supply or induce a
constant level of current in all sample vessels passing through it.
This simplifies operation, whilst still allowing thermal cycling of
each individual sample as it progresses between sites.
[0015] The apparatus may additionally comprise control means,
preferably automatable, by which the supply of current at the
temperature control sites, and/or the temperature of the samples,
may be monitored and/or controlled. Conventional equipment may be
used to perform such tasks.
[0016] The apparatus may comprise additional processing sites
having equipment suitable for processing steps such as sample or
reagent loading or sample monitoring. At some of these processing
sites, conventional apparatus such as heating blocks, ovens, fluid
baths, hot air blowers, fans and the like may, although this is not
normally necessary, be used to provide additional heating and/or
cooling steps for samples passing through.
[0017] One or more of the additional processing sites may be for
monitoring the composition of samples passing through the site
and/or the progress of reactions occurring in the samples, for
example by monitoring the nature and/or level of a target
amplification product in the samples. Reagents in the samples may
be labelled for instance with coloured or fluorescent labels, the
presence of which may be detected at a monitoring site by the
application of suitable radiation. In such cases each sample may be
contained in a vessel at least a portion of which is transparent or
translucent, to allow any applied radiation to reach the samples
and their condition to be appropriately monitored. In this context,
"transparent" and "translucent" mean in respect to any detectable
signal by which the properties of a sample may be monitored--such
signals include, for instance, visible or ultraviolet light,
fluorescence and radioactivity.
[0018] Conventional detection apparatus may be used at a monitoring
site to recognise detectable signals emitted from samples. Such
detection apparatus may for instance comprise means for detecting
the absorption or emission of radiation (eg, visible or ultraviolet
light, fluorescence. radioactivity) by a sample, and/or means for
stimulating such emission, examples being light meters or
luminometers. Reaction monitoring can be efficient, accurate and
continuous throughout the reaction, and samples can be monitored
individually as they pass through the monitoring site.
[0019] Other functions may be carried out at the additional
processing sites. For example, sample vessels may be loaded with
desired reagents at a loading site, and sealed shut at a downstream
site. There may also be sample preparation and/or processing sites,
for instance washing stations or sites at which further samples or
reagents are introduced into sample vessels.
[0020] The conveyor means of the apparatus may comprise
conventional means such as rollers, tracks and conveyor belts, the
exact form depending on the number and nature of the samples and
the way in which they are arranged and supported. The samples can
suitably be contained in vessels such as those described in
WO-98/24548, which comprise electrically conducting materials (in
particular polymers) that heat when an electric current passes
through them. Current may then be supplied to, or induced in, the
vessels as they pass through a site, so as to cause a desired
temperature change.
[0021] However. when using the apparatus of the invention, the
samples are preferably provided on or in a (preferably elongate)
sample support which comprises a succession of separate sample
vessels arranged sequentially one behind the next and is adapted to
be conveyed continuously through a series of processing sites, the
support comprising an electrically conducting material which heats
when an electric current passes through it.
[0022] The sample vessels are preferably separate from one another
and individually sealable and/or isolatable. They may be provided
on or in the support in a linear, or substantially linear.
arrangement. or at least in a non-circular arrangement. The support
may thus preferably be used to allow each sample vessel to reach a
given processing (which includes temperature control) site at a
different time to its adjacent vessel(s) in the succession, and
each vessel may at any given time occupy a different site, and/or
be held at a different temperature. to its adjacent vessel(s). The
support should be continuous over the area supporting the sample
vessels.
[0023] A second aspect of the invention provides such a sample
support, for use with apparatus according to the first aspect.
[0024] The electrically conducting material of the support may be a
metal such as aluminium or copper but is preferably a plastics
material. Electrically conducting polymers, for use in this way,
are known in the art and may be obtained for example from Caliente
Systems Inc. of Newark, USA. Other examples of such polymers are
disclosed for instance in U.S. Pat. Nos. 5,106,540 and 5,106,538.
Suitable conducting polymers can provide temperatures of up to
300.degree. C., ideal for use in PCR processes.
[0025] The electrically conducting plastics material may in
particular be a polymer loaded with an electrically conducting
material. Such conductor-loaded materials are available for
instance from the French company RTP. A polymer, typically a
thermosetting polymer resin such as a polyethylene, polypropylene,
polycarbonate or nylon polymer, may contain embedded in it elements
of an electrically conducting material such as carbon (usually in
the form of fibres) or a metal (copper, for example). These
elements may constitute between say 1 and 50% w/w or higher of the
electrically conducting plastics material.
[0026] An advantage of such polymers is their ability to heat
rapidly. The heating rate depends upon the precise nature of the
polymer, its dimensions and the amount of current applied.
Preferably the polymer has a high resistivity for example in excess
of 1000 ohm.cm. Its temperature can be readily controlled by
controlling the amount of electric current passing through it,
allowing it to be held at a desired temperature for a desired
period of time. The transition rate between temperatures can
similarly be controlled. Moreover, relatively rapid cooling can
also be assured because of the low thermal mass of the polymer.
[0027] The use of such polymers in the construction of sample
vessels, for instance for PCR processing, is described in
WO-98/24548. The polymers may be injection moulded and may
therefore be used directly to form sample vessels and their parts.
Thus, in a sample support according to the invention, an
electrically conducting material preferably plastics, may form part
of or be integral with each sample vessel. Suitably, a sample
vessel or support may be made from another polymer such as
polypropylene which may be moulded with the conducting polymer,
allowing the vessel or support to incorporate separate elements of
the conducting polymer in desired locations. For example, each
sample vessel ideally incorporates one or more electrically
conducting element(s) which are separate from those of adjacent
vessels, to allow individual temperature control for each
vessel.
[0028] Alternatively the support, incorporating the sample vessels,
may be formed from (for instance by injection moulding or
extrusion), or include a layer of, an electrically conducting
material. In this case the support preferably comprises a
succession of electrically isolatable regions corresponding to the
positions of individual sample vessels, again to allow for
independent temperature control. This can be achieved for instance
by providing appropriately positioned electrically insulating
elements (which may include apertures) in the support.
Alternatively the provision of a separate electrode pair for each
sample vessel may allow the supply of a localised current to each
such vessel.
[0029] As a yet further alternative (again as described in
WO-98/24548), an internal surface of each sample vessel may be
coated with the conducting material, for example by a lamination
and/or deposition technique. A conducting plastics material may
suitably be provided in the form of a sheet material or film, for
example of from 0.01 to 10 mm, preferably from 0.1 to 0.3 mm,
thick. A metal conductor may be provided in the form of a foil or
an electrolytically deposited coating of similar thickness.
[0030] In another alternative, an electrically conducting element
is provided in close proximity to, ideally in contact with, each
sample vessel. Suitable arrangements include a sheath of a
conducting material around the sample vessel. Again, the material
is preferably an electrically conducting polymer.
[0031] Electrically conducting plastics materials of the type
described above tend to emit heat when electric current passes
through them, and so may be used to cause a local temperature
change in samples with which they come into contact.
[0032] The use of electrically conducting materials, in particular
plastics materials, in accordance with the present invention allows
a large number of sample-containing vessels to be processed
sequentially and effectively continuously, since each vessel may be
separately supplied with electric current so as independently to
control the temperature of the sample it contains. At the same
time, the incorporation of such temperature control means into the
fabric of the vessel itself can allow relatively simple and compact
sample supports and processing apparatus to be achieved.
[0033] All manner of conventional reaction vessels may be linked
together appropriately, or produced in continuous form, to provide
a sample support of use in the apparatus of the present invention.
Sample vessels in the form of reaction wells may be formed in for
instance a flexible strip by pressing or moulding.
[0034] Thus, a sample support according to the invention may
comprise a strip of a suitably flexible (preferably plastics)
material, on which a succession of sample vessels is mounted or in
which a succession of such vessels is formed. The flexible strip
may itself be formed from, or incorporate (for instance as a
laminate) an electrically conducting material as described
above.
[0035] More preferably, however, the sample support comprises a
series of reaction "units", each of which provides one or more
sample vessels, and which are preferably linked together as a chain
so as to be conveyable sequentially through processing sites.
Examples of such reaction units. although not in linked form, are
described for instance in WO-98/09728 and by Findlay et al in
"Automated Closed-Vessel System for in Vitro Diagnostics Based on
Polymerase Chain Reaction", Clinical Chemistry,39, no. 9, 1993, pp
1927-1933.
[0036] It is possible to utilise a linked chain of reaction vessels
or units because the means for heating each of them (the
electrically conducting material) allows more selective and
localised heating of individual samples, even those which are
adjacent one another in the succession. In turn. the ability to
link a succession of sample vessels or reaction units can greatly
increase processing efficiency, reduce the size and complexity of
processing apparatus and facilitate automation.
[0037] Each reaction unit may for example have the approximate size
and shape of a credit card. The units may be mounted on the sample
support or, conveniently, they may be produced in the form of a
chain of linked units, the chain ideally having sufficient
flexibility to be stored as a roll or as a finned stack. Prior art
reaction systems would have batch processed such units (as
described by Findlay et al, supra), or would have thermally cycled
each unit whilst keeping it stationary at a single processing site;
the present invention allows the units to be processed
continuously, as produced.
[0038] The sample support of the invention preferably comprises
more than three or more than five sequentially arranged sample
vessels, more preferably ten or more, most preferably at least
twenty or fifty or a hundred or more. The vessels may be arranged
in an array, for instance in pairs or in larger groups, so that for
instance two adjacent vessels reach a processing site
simultaneously, another two following behind and another two behind
them, etc.. Suitably the vessels are in the form of capillary
tubes.
[0039] Preferably at least a portion of each vessel is transparent
or translucent to assist in the monitoring of a sample contained in
it.
[0040] The sample support preferably comprises electrical contacts
(for instance, at an edge of the support, and/or provided in each
sample vessel or reaction unit) to facilitate the supply of current
to the electrically conducting material as the support passes
through an appropriate processing site. Alternatively, an electric
current may be induced in the conducting material for example by
exposing it, in use, to suitable electrical or magnetic fields.
Ideally the support and sample vessels are arranged so that each
vessel or at least a set of vessels, may be individually supplied
with current, allowing its temperature to be controlled
independently of other vessels on the support.
[0041] The vessels of the sample support, and/or reaction units
containing them, may be labelled to identify them during
processing, for instance with microchips holding relevant
information. The vessels may be pre-loaded with one or more
reagents, in particular freeze dried, frozen or stabilised
reagents, in conventional fashion. Alternatively reagents may be
dispensed into the vessels at an in-line pipetting station provided
in apparatus according to the invention.
[0042] The sample support, and/or each of the sample vessels or
reaction units it comprises, is preferably designed to be
disposable after use.
[0043] The support may comprise an electrically conducting layer
and a facing layer with one or more reagent wells defined between
them, as described for instance (although not in continuous form)
in co-pending UK patent application number 9922971.8. Such sample
vessels may be filled with appropriate reagents and then sealed
prior to undergoing thermal cycling.
[0044] Using apparatus according to the first aspect of the
invention, sample vessels on a support according to the second
aspect may be filled and/or sealed at processing sites upstream of
the temperature control sites and optional monitoring sites. As
with other aspects of the use of the apparatus, these steps may be
partially or fully automated.
[0045] Apparatus according to the invention may therefore comprise,
upstream of the temperature control sites, means for loading
reagents into a succession of sample vessels, preferably provided
on or in a sample support, and/or means for sealing loaded sample
vessels. It also preferably comprises means for producing a sample
support of the type described above and means for conveying the
so-produced sample support to downstream processing sites.
[0046] A third aspect of the present invention provides a method
for carrying out a thermal cycling reaction, which involves using
apparatus according to the first aspect, and/or a sample support
according to the second, to convey a succession of samples through
a series of sequentially arranged temperature control sites, at
each of which sites an electric current is supplied to, or induced
in, a sample-containing vessel passing through them so as to induce
temperature changes in the sample.
[0047] In such a method, the samples are preferably conveyed
continuously through the temperature control sites. The thermal
cycling reaction is suitably part of an amplification reaction, in
particular a PCR reaction.
[0048] The method is preferably at least partially automated, for
instance under computer control. It can enable high throughput
testing, which is especially desirable for diagnostic methods such
as the DNA amplification of pathogens or other contaminants
(including genetic pollution) in for instance the air, body fluids,
foodstuffs and the like.
[0049] The method may be particularly useful in the online
monitoring of environmental conditions, for instance in a storage
atmosphere, a reaction mixture, a water or food supply, a
manufactured product or by-product, a waste outlet or even in body
fluids in vivo.
[0050] Samples may be continually extracted from the environment of
interest and subjected successively, using the method of the
invention, to a diagnostic process involving thermal cycling. As
the samples pass through monitoring sites, time-dependent profiles
of their composition may be acquired.
[0051] At one or more additional processing sites, samples may
accordingly be acquired, and/or loaded into vessels and/or
monitored, as described above in connection with the apparatus of
the invention.
[0052] According to a fourth aspect, the present invention provides
a method for producing a sample support according to the second
aspect, the method comprising forming (for instance by pressing) a
succession of reaction wells in a flexible strip comprising an
electrically conducting (preferably plastics) material which heats
when an electric current passes through it. This method may include
providing one or more of the reaction wells with one or more
appropriately positioned electrical contacts. It may also include
pre-loading one or more of the reaction wells with a desired
reagent or reagents.
[0053] Again, the flexible strip may be made of an electrically
conducting material, or it may incorporate a layer of such a
material.
[0054] The method of the fourth aspect of the invention may be
incorporated into that of the third aspect.
[0055] According to fifth and sixth aspects of the invention, there
are provided (a) apparatus according to the first aspect in
combination with a sample support according to the second aspect,
and (b) the use of a sample support according to the second aspect
in a method according to the third.
[0056] The present invention will now be described in more detail
with reference to the accompanying illustrative drawings, of
which:
[0057] FIG. 1 illustrates a method and apparatus in accordance with
the invention;
[0058] FIGS. 2 and 3 illustrate alternative methods and apparatus
according to the invention;
[0059] FIGS. 4 and 5 are vertical longitudinal sections through
sample supports for use in the methods and apparatus of FIGS. 1, 2
or 3;
[0060] FIG. 6 is a horizontal section through the sample support of
FIG. 5;
[0061] FIG. 7 is a plan view of a sample support passing through
apparatus in accordance with the invention;
[0062] FIG. 8 is a vertical section through the FIG. 7 arrangement;
and
[0063] FIGS. 9 and 10 are a plan view and vertical section
respectively of a sample support passing through an alternative
apparatus according to the invention.
[0064] All drawings are schematic.
[0065] Referring firstly to FIG. 1, the method illustrated involves
conveying a succession of samples, on a continuous support, through
a series of sequentially arranged processing sites 1-7 in the
direction shown by the arrows. In this case sites 3-6 are
temperature control sites at which the samples are thermally cycled
between desired temperatures. The additional processing sites are
for (1) loading samples into sample vessels, (2) sealing the open
ends of the vessels and (7) monitoring the progress of reactions in
the samples, and/or the sample composition (for instance in an
assay for detecting a target material in the sample) by irradiating
the samples and detecting light emitted by appropriately labelled
reagents. [Alternatively one or more complete thermal cycles of
heating and cooling can be carried out at any of sites 3, 4, 5
and/or 6]. Conventional apparatus, preferably automated, is used at
the seven sites to effect the necessary processing steps.
[0066] Typically, apparatus according to the invention may comprise
many more processing sites than the seven shown schematically in
FIG. 1. For instance, it may comprise 150 or more temperature
control sites in order to carry out a typical PCR reaction of three
or more steps.
[0067] In the FIG. 1 system, the samples are contained in
disposable reaction units of the general form disclosed in for
instance WO-98/09728, or by Findlay et al (supra), or in co-pending
UK patent application number 9922971.8 (see FIG. 4). Each unit
provides an array of reaction "wells", which can be loaded at site
1 with the desired reagents and sealed shut at site 2. A continuous
chain 8 of such units, linked together by flexible plastics
"bridges", is stored on a roll 9 and from there is fed through the
processing sites 1-7. Conventional drive means (not shown) are used
to move the chain 8 through the apparatus automatically.
[0068] The FIG. 2 system is identical to that of FIG. 1, except
that the chain 8 of reaction units is stored as a fanned stack
10.
[0069] In the alternative system of FIG. 3, a chain 11 of reaction
units is manufactured at an additional site 12 upstream of the
processing sites 1-7, and from thence fed directly through the
apparatus to allow the desired thermal cycling reactions to take
place.
[0070] Sample supports of use in the FIGS. 1, 2 and 3 systems are
shown in FIGS. 4 and 5. That of FIG. 4 is in the form of an
elongate flexible strip 20 in which a succession of generally
tubular sample wells 21 has been punched using a conventional die
and tube former. The strip is made from an electrically conducting
polymer, of the type described above, which heats when electric
current passes through it. Each sample well is provided with
electrical contacts 22 to enable current to be supplied to it at
appropriate stages in processing. The wells may be pre-loaded with
for instance dried or frozen reagents, as shown at 23.
[0071] A method in accordance with the invention may include the
steps of punching out the sample wells 21 in a blank polymer strip,
introducing the electrical contacts 22, loading the desired
reagents into the wells, sealing the loaded wells shut (for
instance, by heat sealing, or by means of an adhesive or plug) and
then conveying the thus-formed succession of samples through a
series of temperature control sites and optional additional
processing sites such as monitoring sites. Thus the entire process
may be conducted continuously, and lends itself well to complete
automation.
[0072] The FIGS. 5 and 6 sample support comprises a series of
approximately credit card sized reaction "units" 30 provided in a
flexible strip generally labelled 31. The strip comprises a thin
aluminium foil backing layer 32, a polycarbonate spacing layer 33
adhered to the backing layer by an adhesive layer 34 and an
optically transparent polycarbonate facing layer 35 adhered to the
spacing layer by adhesive 36. In each unit, the spacing layer 33 is
provided with an array of holes 37 (in this case, six) which define
sample wells. The holes 37 communicate with a channel 38 and an
inlet 39 (see FIG. 6; omitted from FIG. 5 for clarity) through
which reagents may be introduced into the sample wells. The inlet
is sealed shut prior to carrying out thermal cycling reactions on
the enclosed samples. Loading and sealing may be effected by
methods described in for instance WO-98/09728, Findlay et al
(supra), or co-pending UK patent application number 9922971.8.
[0073] The presence of the thermally conductive aluminium layer 32
reduces the time needed to heat or cool samples in the unit to
desired temperatures. Electrodes 40 are provided on the strip 31
adjacent each "unit" (see FIG. 6).
[0074] There may be any number of sample wells provided in each
unit, arranged in any appropriate manner. The wells may be
pre-loaded with desired reagents.
[0075] The strip 31 is provided with a regularly spaced succession
of engageable driving means, in this case sprocket holes 41 (FIG.
6), via which it may be driven through a succession of processing
sites. It is scored along the lines 42 between adjacent units, to
increase its flexibility.
[0076] Alternative sample supports in accordance with the invention
may comprise a flexible backing strip corresponding for instance to
the foil backing layer 32 of FIGS. 5 and 6, onto which is mounted a
series of reaction units incorporating the facing and spacing
layers 35 and 33. The backing strip could be made of any
electrically conducting material, in particular an electrically
conducting polymer.
[0077] As a further alternative, the conducting layer may be
omitted and instead electrically conducting elements incorporated
separately into each sample well. These could take the form of
appropriately placed regions of an electrically conducting
polymer.
[0078] FIG. 7 illustrates how a chain of individual PCR reaction
vessels (tubes 43), linked together in any appropriate manner, may
be conveyed through a series of processing sites 44 in accordance
with the present invention. At each site a pair of moveable
actuators 45 is arranged to apply a magnetic field to, and hence
induce a current in, conducting elements present in the tubes as
they pass through the site.
[0079] Each tube 43 (see FIG. 8) is a two-part injection moulding
formed primarily from polypropylene but incorporating a shaped
outer layer 46 of an electrically conducting polymer. This outer
layer heats when current is induced in it by the actuators 45, thus
supplying heat to the contents of the tube.
[0080] The tube 43 also has a plug 47 by which its open end is
sealed after sample loading.
[0081] In the alternative system illustrated in FIGS. 9 and 10, a
chain of PCR tubes 48 is conveyed through apparatus according to
the invention by pairs of "pinch rollers" 49. The rollers are made
of an electrically conducting material such as steel and are
mounted so that, in use, they form an electrical contact with a
conducting polymer outer layer 50 (see FIG. 10) of each tube as it
passes them. This contact may be via appropriately positioned
brushes or the like, not shown in the figures.
* * * * *